Name: rearing and injection of manduca sexta larvae to assess bacterial virulence
Uploaded: 2012-12-11T12:00:00
Description: Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

The authors wish to thank past members of the Goodrich-Blair lab: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard, and Youngjin Park for their contributions to the development of this protocol. This work was funded by the National Science Foundation grant IOS-0950873 and the National Institutes of Health NRSA fellowship FAI084441Z.

1. Insect Egg Sterilization and Rearing
<ol>
	<li>Prepare diet by first autoclaving 15 g of the provided agar in 900-1,000 ml H2O. Immediately after autoclaving, mix with 166 g wheat germ diet and blend well in a laboratory blender. Pour into a dish (or dishes) to cool, then transfer diet to aluminum foil, wrap tightly, and store at 4 &deg;C.</li>
	<li>Upon arrival, sterilize M. sexta eggs with 250 ml of 0.6% bleach solution for 2-3 min in a glass filter holder and vacuum flask apparatus with a 90 m...

<ol>
	<li>Bintrim, S. B., Ensign, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+inactivation+of+genes+encoding+the+crystalline+inclusion+proteins+of+Photorhabdus+luminescens+results+in+mutants+with+pleiotropic+phenotypes.">Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes.</a> J. Bacteriol. 180, 1261-1269 (1998).</li><li>Schesser, J. H., Kramer, K. J., Bulla, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioassay+for+homogeneous+parasporal+crystal+of+Bacillus+thuringiensis+using+the+tobacco+hornworm,+Manduca+sexta.">Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hornworm, Manduca sexta.</a> Appl. Environ. Microbiol. 33, 878-880 (1977).</li><li>P&#233;chy-Tarr, M., Bruck, D. J., Maurhofer, M., Fischer, E., Vogne, C., Henkels, M. D., Donahue, K. M., Grunder, J., Loper, J. E., Keel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+a+novel+gene+cluster+encoding+an+insect+toxin+in+plant-associated+strains+of+Pseudomonas+fluorescens.">Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens.</a> Environ. Microbiol. 10, 2368-2386 (2008).</li><li>Nu&#241;ez-Valdez, M. E., Calder&#243;n, M. A., Aranda, E., Hern&#225;ndez, L., Ram&#237;rez-Gama, R. M., Lina, L., Rodr&#237;guez-Segura, Z., Guti&#233;rrez Mdel, C., Villalobos, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+putative+Mexican+strain+of+Serratia+entomophila+pathogenic+against+root-damaging+larvae+of+Scarabaeidae+(Coleoptera).">Identification of a putative Mexican strain of Serratia entomophila pathogenic against root-damaging larvae of Scarabaeidae (Coleoptera).</a> Appl. Environ. Microbiol. 74, 802-810 (2008).</li><li>Forst, S. A., Tabatabai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+histidine+kinase,+EnvZ,+in+the+production+of+outer+membrane+proteins+in+the+symbiotic-pathogenic+bacterium+Xenorhabdus+nematophilus.">Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.</a> Appl. Environ. Microbiol. 63, 962-968 (1997).</li><li>Kanost, M. R., Jiang, H., Yu, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+a+lepidopteran+insect,+Manduca+sexta.">Innate immune responses of a lepidopteran insect, Manduca sexta.</a> Immunol. Rev. 198, 97-105 (2004).</li><li>Yu, X. Q., Zhu, Y. F., Ma, C., Fabrick, J. A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+proteins+in+Manduca+sexta+plasma.">Pattern recognition proteins in Manduca sexta plasma.</a> Insect Biochem. Mol. Biol. 32, 1287-1293 (2002).</li><li>Eleftherianos, I., ffrench-Constant, R. H., Clarke, D. J., Dowling, A. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+immune+response+to+the+entomopathogen+Photorhabdus.">Dissecting the immune response to the entomopathogen Photorhabdus.</a> Trends Microbiol. 18, 552-560 (2010).</li><li>Herbert, E. E., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friend+and+foe:+the+two+faces+of+Xenorhabdus+nematophila.">Friend and foe: the two faces of Xenorhabdus nematophila.</a> Nat. Rev. Microbiol. 5, 634-646 (2007).</li><li>D'Argenio, D. A., Gallagher, L. A., Berg, C. A., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+host+for+Pseudomonas+aeruginosa+Infection.">Drosophila as a model host for Pseudomonas aeruginosa Infection.</a> J. Bacteriol. 183, 1466-1471 (2001).</li><li>Waterfield, N., Dowling, A., Sharma, S., Daborn, P. J., Potter, U., Ffrench-Constant, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+toxicity+of+Photorhabdus+luminescens+W14+toxin+complexes+in+Escherichia+coli.">Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.</a> Appl. Environ. Microbiol. 67, 5017-5024 (2001).</li><li>Park, Y., Herbert, E. E., Cowles, C. E., Cowles, K. N., Menard, M. L., Orchard, S. S., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+variation+in+Xenorhabdus+nematophila+virulence+and+suppression+of+Manduca+sexta+immunity.">Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity.</a> Cell. Microbiol. 9, 645-656 (2007).</li><li>Park, Y., Kim, Y., Putnam, S. M., Stanley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterium+Xenorhabdus+nematophilus+depresses+nodulation+reactions+to+infection+by+inhibiting+eicosanoid+biosynthesis+in+tobacco+hornworms,+Manduca+sexta.">The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 52, 71-80 (2003).</li><li>Cowles, K. N., Cowles, C. E., Richards, G. R., Martens, E. C., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+regulator+Lrp+contributes+to+mutualism,+pathogenesis+and+phenotypic+variation+in+the+bacterium+Xenorhabdus+nematophila.">The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila.</a> Cell. Microbiol. 9, 1311-1323 (2007).</li><li>Cowles, K. N., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+activity+of+a+Xenorhabdus+nematophila+haemolysin+required+for+full+virulence+towards+Manduca+sexta+insects.">Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects.</a> Cell. Microbiol. 7, 209-219 (2005).</li><li>Goodrich-Blair, H., Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutualism+and+pathogenesis+in+Xenorhabdus+and+Photorhabdus:+two+roads+to+the+same+destination.">Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination.</a> Mol. Microbiol. 64, 260-268 (2007).</li><li>Eleftherianos, I., Baldwin, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+modulation+of+immunity:+changes+within+the+feeding+period+of+the+fifth+larval+stage+in+the+defence+reactions+of+Manduca+sexta+to+infection+by+Photorhabdus.">Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.</a> J. Insect Physiol. 54, 309-318 (2008).</li><li>Kavanagh, K., Reeves, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+potential+of+insects+for+in+vivo+pathogenicity+testing+of+microbial+pathogens.">Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens.</a> FEMS Microbiol. Rev. 28, 101-112 (2004).</li></ol>

The direct injection of M. sexta larvae with entomopathogenic bacteria, as described here, serves as a simple and effective means to analyze bacterial virulence. The method is also highly adaptable to suit different experimental subjects and/or conditions. Bacteria can be prepared in various ways prior to injection. In the case of X. nematophila, wild type cells grown in nutrient-rich Luria-Bertani (LB) medium to mid-log phase are typically the most virulent, killing most or all insects within 30 hr sub...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>90 mm filter paper</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass filter holder</td>
			<td>Millipore</td>
			<td>XX1004700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Manduca sexta eggs</td>
			<td>Carolina Biological Supply</td>
			<td>143880</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gypsy Moth Diet + agar</td>
			<td>MP Biomedicals</td>
			<td>0296029301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5.5 oz. plastic containers and lids</td>
			<td>Solo Cup Company</td>
			<td>URC55-0090 Pl4-0090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 oz. plastic containers and lids</td>
			<td>DART Container Corporation</td>
			<td>100PC 100PCL25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>137 mm NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4, pH 7.4</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>80208</td>
			<td>30 gauge, 0.375" length, point style 2</td>
		</tr>
	</tbody>
</table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

A representative example of an insect mortality assay is depicted in Figure 3. In this experiment, insects were injected with approximately 50 colony forming units (CFU) of either wild type (ATCC19061) or an attenuated mutant strain (lrp13) of Xenorhabdus nematophila grown to mid-log phase (n=6 insects per strain). Insects were observed for approximately 72 hr, and the percent of injected insects still alive at each timepoint recorded. In this case, the attenuated strain exhi...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...